Melanoma cancer database and data processing
GSE46517, GSE52882 and GSE54493 datasets containing mRNA expression profiles were downloaded from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). GSE46517 dataset included 5 normal tissue samples, 5 primary melanoma tissue samples and 5 metastatic melanoma tissue samples; GSE52882 included 3 DMSO treated A375 cells, 3 vemurafenib treated A375 cells, 3 DMSO treated A2058 cells and 3 vemurafenib treated A2058 cells; GSE54493 included 2 mock transfected SK-Mel-147 cells, 2 miR-638 OE transfected SK-Mel-147 cells, and 2 miR-638 knockdown (KD) transfected SK-Mel-147 cells.
Identification of DEGs
Firstly, the experiment was divided into six groups: 1) 5 normal tissue samples vs 5 primary melanoma tissue samples in GSE46517 dataset ; 2) 5 normal tissue samples vs 5 metastatic melanoma tissue samples in GSE46517 dataset ; 3) 3 DMSO treated A375 cells vs 3 vemurafenib treated A375 cells in GSE52882 dataset; 4) 3 DMSO treated A2058 cells and 3 vemurafenib treated A2058 cells in GSE52882 dataset; 5) 2 mock transfected SK-Mel-147 cells vs 2 miR-638 OE transfected SK-Mel-147 cells in GSE54493 dataset; 6) mock transfected SK-Mel-147 cells vs 2 miR-638 KD transfected SK-Mel-147 cells in GSE54493 dataset. The DEGs were calculated using the “GEO2R” online tool (http://www.ncbi.nlm.nih.gov/geo/geo2r). The DEGs of the database with an absolute Log2 fold change (FC) > 1 and P value < 0.05 were considered for subsequent analysis.
Functional enrichment analysis
The GO enrichment was used to analyze DEGs at the functional level with DAVID (http://david.ncifcrf.gov/,version 6.8). DAVID is a comprehensive database of functional annotation tools for connecting functional terms with gene lists using a clustering algorithm. In order to elucidate the functional profiles of the DEGs, we used DAVID to obtain the enriched biological process (BP), cellular component (CC), molecular function (MF) and KEGG pathway. P < 0.05 was considered statistically significant.
PPI network analysis
The STRING database (http://string-db.org/) was used to identify the interactions between proteins encoded by DEGs based on experimental data, databases, text mining, and predictive bioinformatics data. A combined score of > 0.4 was set as threshold value. PPI networks were constructed with Cytoscape software. The plug-in MCODE was used to screen the modules of PPI network in Cytoscape software. MCODE score ≥ 3 and number of nodes ≥ 3 were considered as threshold value.
Expression and survival analysis of IFITM1 and LTBP4
TCGAportal (http://www.tcgaportal.org) and GEPIA (http://gepia.cancer-pku.cn/) were used to investigate survival probability and the expression of IFITM1 and LTBP4 in tumor tissues and corresponding para-carcinoma tissues.
Cells and surgical specimens
Seven melanoma cell lines used in this study. A101D, 451Lu, SK-MEL-1, VMM5A, G-361, A375, and MeWo cells and control cell (HaCaT cell) were purchased from the Cell Culture Collection of the Chinese Academy of Sciences (Beijing, China) and cultured in DMEM medium containing 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 mg/mL streptomycin, and 100 U/mL penicillin in a humidified incubator containing 5% CO2 at 37°C for 2-3 days. Specimens of melanoma tissues and adjacent noncancerous tissues were collected from 76 patients with melanoma between October 2018 and October 2019, following surgical resection at Tianjin Medical University Eye Hospital. All experiments were approved by the Medical Ethics Committee of Tianjin Medical University Eye Hospital and written informed consent documents were signed by all of the patients. Table 1 lists the clinical characteristics of the enrolled patients.
LTBP4 gene silencing and OE and cell transfection
Two different shRNAs (shRNA-1 and shRNA-2; purchased from GeneChem, Shanghai, China) were used to target the LTBP4 gene KD. A nonsilencing shRNA (NC) was used as control (GeneChem). LTBP4 expressing plasmids (constructed using a pcDNA 3.1 vector) (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to induce LTBP4 OE. Cells were transfected with 10 nM of shRNAs or pcDNA3.1 when cells reached 30-50% confluence. After 8 hours of transfection using Lipofectamine-2000 (Life Technologies) according to the manufacturer’s protocol, cells were returned to normal medium in the incubator. When cells reached 85% confluence, cell supernatant, protein, and RNA were collected for later experiments.
YAP gene or MST1 genes OE and cell transfection
YAP expressing plasmids or MST1 expressing plasmids (constructed using a pcDNA 3.1 vector) (Invitrogen; Thermo Fisher Scientific, Inc) to induce YAP OE or MST1 OE. SK-MEL-1 and A375 cells were transfected with 10 nM of pcDNA3.1 when cells reached 30-50% confluence. After 8 hours of transfection using Lipofectamine-2000 (Life Technologies) according to the manufacturer’s protocol.
This experiment was divided into the following four groups:1) SK-MEL-1 or A375 cells without any treatment was viewed as CTRL group; 2) SK-MEL-1 or A375 cells transfected with LTBP4 OE was viewed as LTBP4-OE group; 3) SK-MEL-1 or A375 cells co-transfected with LTBP4 OE and YAP OE was viewed as LTBP4-OE+YAP-OE group; 4) SK-MEL-1 or A375 cells co-transfected with LTBP4 OE and MST1 OE was viewed as LTBP4-OE+MST1-OE group. Cell proliferation, apoptosis, migration and invasion in above groups were measured using CCK-8, colony formation, flow cytometry, transwell and wound healing assays.
Cell viability assay
Cells were transfected with shRNAs or pcDNA3.1 for 48 h to detect cell viability according to CCK-8 kit instructions (Beyotime, Shanghai, China). Transfected cells (5,000 cells per well) were incubated in a cell incubator for 0.5-4 h and tested at 0, 6, 12, 24, 48 and 72 h, respectively, using a microplate reader. Absorbance was measured at 450 nm.
For the colony formation assay, 600 cells per group were plated in triplicates in a six-well plate. Cells were allowed to grow till visible colonies appeared, stained with crystal violet (Beyotime), washed with PBS, and counted.
Flow cytometry analysis
For apoptosis measurements, the percentages of apoptotic cells were determined by flow cytometry using the Annexin V-FITC/PI cell apoptosis detection kit (Promega) according to the manufacturer’s instructions.
After transfection with the different plasmids for 24 h, cells were seeded in a 24-well plate and grown to confluence. The confluent monolayer of cells was wounded using a standard 200 µL pipette tip and then washed three times to remove the non-adherent cells. Wounds were monitored and photographed at the time the scrape was created and 24 h later. Cell migration capacity was calculated according to the width of the wounds at 0 h and 24 h. The migration rate is described as a percentage of the migration observed in the control group under an inverted microscope (Olympus).
Transwell invasion assays
Transwell assays were conducted to detect the invasion capabilities of cells. Cells (4×105/mL) were plated within the top chamber coated with Matrigel membrane. FBS 10% was used as a chemoattractant in the lower chamber. Cells were incubated for 48 hours, and the cells that did not invade through the membrane were removed by a cotton swab. Cells on the lower chamber were then stained with crystal violet and counted under an inverted microscope (Olympus).
In vivo tumorigenicity
Animal experiments were done according to Institutional Animal Care and Use Committee (IACUC) protocol and approved by Tianjin Medical University Animal Center for Use and Care of Animals. The establishment of the subcutaneous melanoma tumor model was treated by subcutaneous injection of SK-MEL-1 cells. A total of 12 4-week-old female BALB/C nude mice (Tianjin Medical University Animal Center, Tianjin, China) were required to establish melanoma model mice for 4 weeks. Mice were sacrificed 30 days after the injection and size of the tumor was measured by vernier caliper and weight of the tumor was measured by electronic balance.
Immunohistochemistry (IHC) staining
LTBP4 expression between melanoma tissues and adjacent noncancerous tissues in patients with melanoma was determined by IHC staining. For IHC, sections were incubated with anti-LTBP4 (1:300 dilution) antibody. LTBP4 staining was scored by two independent pathologists. LTBP4 or cleaved caspase-3 expression between NC group and LTBP4-KD group in tumor tissues of BALB/C nude mice was determined by IHC staining. For IHC, sections were incubated with anti-LTBP4 (1:500 dilution) antibody or anti-cleaved caspase-3 (1:200 dilution) antibody. LTBP4 or cleaved caspase-3 staining was scored by researcher. The scoring system was based on the staining intensity and extent. Staining intensity was classified as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). Staining extent depended on the percentage of positive cells and was divided into 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). According to the staining intensity and the staining extent scores, the IHC result was classified as 0–1, negative (−); 2–4, weakly positive (+); 5–8, moderately positive (++); and 9–12, strongly positive (+++).
Protein lysates were prepared from cells using 500 µL of RIPA buffer with 1 mM phenylmethane sulfonyl fluoride. Samples were subsequently sonicated for 2 minutes and centrifuged. The supernatants were collected and used for protein analysis. Lysates were separated on 8% polyacrylamide gels and transferred onto polyvinylidene fluoride membrane. The membranes were blocked with PBS containing 0.1% Tween-20 (PBST) and 5% nonfat milk (w/v) for 1 hour at room temperature. After they were washed with PBST, the membranes were probed with antibodies overnight at 4°C. Antibody against LTBP4 was obtained from Shanghai Yu Bo Biotech Co,Ltd (Shanghai, China); antibody against cleaved caspase-3/Ki67/E-cadherin/YAP/MST1/CTGF/Cyr61 and phosph-YAP/MST1 antibody was obtained from Abcam (Cambridge, UK); antibody against MOB1 and phospho-MOB1 were obtained from Cell Signaling Technology, Inc (USA); antibody against Birc5 and phosphor-Birc5 were obtained from CUSABIO engineering co. Ltd (Wuhan, Hubei, China); antibody against β-actin or LaminB were obtained from Beyotime (China). The membranes were washed again with PBST, then horseradish peroxidase-labeled IgG at 1:5,000 dilution was added at room temperature for 1 h, and the blots were developed using enhanced chemiluminescence western blotting reagents. β-actin or LaminB was used as an internal control.
Real-time PCR (RT-PCR)
Real-time PCR was performed on a Step Two Real-Time PCR System (Applied Biosystems) using the comparative Ct quantization method. Real-time PCR Master Mix (Toyobo) was used to detect and quantify the expression level of the target gene. GAPDH was used as an internal control. The primers used were as follows: CTGF, 5'- AGTGCATCCGTACTCCCAAA-3' (F) and 5'- CCGTCGGTACATACTCCACA-3' (R); Cyr61, 5'- GCAGCGTTTCCCTTCTACAG-3' (F) and 5'- ATGAGTCCCATCACCCACAC-3' (R); Birc5, 5'- AACAGTGGCTGCTTCTCTCT-3' (F) and 5'- GCCTTCTTCCTCCCTCACTT-3' (R); β-actin, 5'- ACTCTTCCAGCCTTCCTTCC-3' (F), 5'-CAATGCCAGGGTACATGGTG-3' (R); YAP, 5'-TCCACCAGTGCAGCAGAATA-3' (F) and 5'- TTGGGTCTAGCCAAGAGGTG-3' (R); MST1, 5'-TCCCAGTAGCCAAGATGGTG-3' (F) and 5'- TGGAGGCACCACATACCATT-3' (R); LTBP4, 5'- CGACATGCCAGACTTTGAGG-3' (F) and 5'- ACCAGCATAGCTTCCACCTT-3' (R).
Statistical analyses were performed using SPSS 19.0 (IBM, Armonk, NY, USA). Correlations between LTBP4 expression and the clinicopathological variables were analyzed using the Pearson χ2 analysis. The average value of LTBP4 > 1.925 as high expression. Survival was analyzed using the Kaplan-Meier method, and differences were evaluated using the log-rank test. The Cox proportional hazards model was used for univariate analysis to examine the potential prognostic value of different variables on OS. Data were evaluated using ANOVA with LSD test for multiple comparisons and Student’s t test between two groups. P < 0.05 was considered to indicate a statistically significant difference.